Smith-Lemli-Optiz/RSH syndrome (SLOS) is a genetic disorder that affects the development of children both before and after birth. SLOS affects about 1:20,000 individuals. The syndrome was first described in 1964 in three boys with poor growth, developmental delay, and a common pattern of congenital malformations including cleft palate, genital malformations, and polydactyly (extra fingers and toes). In 1993 scientists discovered that children with SLOS are unable to make sufficient cholesterol.
The Smith-Lemli-Opitz syndrome (SLOS) is caused by impaired activity of the enzyme 3β-hydroxsterol, Δ7-reductase (7DHCR)(Irons et al., Lancet 341: 1414, 1993; Tint et al., N Engl J Med 330: 107–113, 1994), which is involved in the enzymatic conversion of 7-dehydrocholesterol to cholesterol. in one of two proposed routes of cholesterol biosynthesis (Scheme 1). The defect in 7DHCR results in an abnormal accumulation of 7- and 8-dehydrocholesterol (1 and 2). More than 60 enzyme mutations have been detected in SLOS-affected individuals (Fitzky et al., Proc Natl Acad Sci USA 95: 8181–8186, 1998; Moebius et al., Proc Natl Acad Sci USA 95: 1899–1902, 1998; Wassif et al., Am J Hum Genet 63: 329–338, 1998).

The challenge in prenatal diagnosis has been the identification of a non-invasive test that involves detection of definitive and SLOS-specific analyte(s), and which can be performed early in pregnancy. Many of the conventional SLOS screening assays involve detecting varying levels of 7-DHC, cholesterol or estriol (E3) or a combination thereof, in chorionic villus (CV) or amniotic fluid samples, each of which are invasive tests and can pose a risk to the fetus. TABLE 1 lists the most common conventional SLOS markers for the screening of SLOS affected fetuses.
TABLE 1EXEMPLARY SLOS MARKERSLow Cholesterol levels in CV biopsies and amniotic fluid.Increased 7-DHC levels (Dehydrocholesterol) in CV and amniotic fluid.Increased 8-DHC levels (dehydrocholesterol II) in CV and amniotic fluid.Low unconjugated estriol levels in serum (non-specific marker)
Since fetal cholesterol is a precursor to estriol (E3), E3 is decreased in SLOS affected pregnancies (Donnai et al., J Med Genet 23: 64–71, 1986; McKeever and Young, J Med Genet 27: 465–466, 1990; Abuelo et al., Am J Med Genet 56: 281–285, 1995; Rossiter et al., Am J Med Genet 56: 272–275, 1995). However, detection of E3 levels is not specific for SLOS-affected pregnancies. Currently, unconjugated serum estriol (uE3) is measured in about 50% of all United States pregnancies as part of the “triple marker screening” for chromosomal aneuploidies and neural tube defects (Palomaki et al., Am J Med Genet 176: 1046–1051, 1997). In 1999, Bradley and co-workers (Bradley et al., Amer. J. Med. Gen. 82:355–358, 1999) published a retrospective study of 26 SLOS pregnancies in which uE3 in serum had been measured and determined that the mean uE3 level was 0.23 of normal median (multiples of the median, MOM). Assaying for low estriol levels in maternal serum MsuE3 along with sonography have also been suggested for diagnosing RSH/SLOS (Kratz, L. E., Kelley, R. I., Amer. J. Med. Gen. 82:376–381, 1999) as well as identifying patients with low maternal urinary levels of estriol (McKeever and Young, 1990) at mid-gestation.
Many SLOS diagnostic methods have been suggested in which the level of 7-DHC is detected due to the increase of this cholesterol precursor in SLOS affected patients. High levels of 7-DHC and 8-DHC have been detected in amniotic fluid and in CV biopsies of SLOS patients (Rossiter, J. P. et. al., Amer. J. Med. Gen. 56:272–275, 1995; Tint, G. S. et. al., Prenat. Diagn. 18:651–658, 1998; Irons, M. B., Tint, G. S., Prenat. Diagn. 18:369–372, 1998; Kratz, L. E., Kelley, R. I., Amer. J. Med. Gen. 82:376–381, 1999). High levels of 7-DHC have also been detected in CV biopsies of SLOS patients as early as the first trimester (Sharp, P. et al., Prenat. Diagn., 17(4): 355–361, 1997). In addition to detection of 7-DHC and 8-DHC, high levels of lathosterol (cholest-7-en-3beta-ol), a 7-DHC precursor, have also been detected in amniotic fluid.
Mills, K. et. al., Pediatric Research 39(5): 816–819 (1996) describe a method for detecting SLOS by determining the ratio of 7-DHC (a cholesterol precursor) to cholesterol in chorionic villus (CV) samples. Mills et al. determined that cholesterol synthesis via 7-DHC occurs in the placenta and/or fetus at 10 weeks of gestation and that prenatal diagnosis by CV biopsy is possible. While this test can detect SLOS early in gestation, CV biopsy is an invasive procedure and is associated with some risk to the fetus and patient.
Recently, it was shown that mid-gestational urine from a SLOS affected pregnancy contains metabolites unsaturated analogs of the compounds estriol (E3) and pregnanetriol (PT). These compounds were suggested to be synthesized or metabolized from fetal 7- or 8-DHC (Shackleton et al., Steroids. 1999a, 64(7): 446–52; Shackleton et al., J. Clin. Endocrinol. Metab. 1999b, 84(3): 1157–9; Shackleton et al., Steroids. 1999c, 64(7): 481–90).
Shackleton et al 1999a, disclose that either 7-DHPT or 8-DHPT of the 3,16,20 and 3,17,20 (triol structures) series was present in the maternal urine of one healthy 35 year-old women carrying a SLOS fetus at 17 weeks gestation. The authors provisionally characterize the SLOS metabolite as 5 pregn-7(or 8-)-ene-3 α,17α, 20α-triol; 5β-pregn-7(or 8-)-ene-3α,16α,20α-triol; 5α-pregn-7(or 8-)-ene-3α,16α,20α-triol; 5α-pregn-7(or 8-)-ene-3α,17α,20α-triol and/or 5α-pregn-7(or 8-)-ene-3β,16α,20α-triol. The authors indicated that the major SLOS metabolite compound is either 5β-pregn-7-ene-3α,17α,20α-triol (7-DHPT) and/or 5β-pregn-8-ene-3α, 17α,20α-triol (8-DHPT). The authors did not indicate if the tentative SLOS metabolite was a mixture of the two epimers or pure 7-DHPT or 8-DHPT and no isolation of the specific compound was attempted. Furthermore, Shackleton et al. 1999a did not show detection of the analytes prior to 17 weeks gestation.
In a separate study of three young infants affected with SLOS, the authors detected a SLOS metabolite(s) and provisionally identified the compound(s) as 3β, 16α-dihydroxy-5,7-pregnadien-20-one; 3β, 16α-dihydroxy-5,8 (or 9-)pregnadien-20-one; homologues of 16α-hydroxy-DHEA, as well as the 7- or 8-epimer of 5β-pregnene-3α,17α,20α-triol (Shackleton et al., Steroids. 1999c, 64(7): 481–90). In short, while these studies narrow the possibilities of the identity of a unique SLOS analyte, they failed to confirm the identification due to the complexity of the mass spectra profile of the biological sample and the lack of appropriate reference compounds.
While these two SLOS specific metabolites were tentatively characterized by Shackleton et al., the actual structures and identification of the two SLOS specific analytes (Δ7 or Δ8) was not determined. Depending upon the sensitivity of the detection system, a detectable amount of these SLOS analyte may be found in normal patients, an assay which only detected the presence of a epimer mixture of these compounds or the wrong epimer without proper controls, could lead to a high frequency of false positives and false negatives, making the assay unpredictable, unreliable and not commercially viable. These risks of false positives and false negatives are further exacerbated when one considers that low levels of SLOS analyte levels are found in affected SLOS individuals, thus necessitating the use of sensitive detection methods such as gas chromatography/mass spectroscopy (GC/MS).
Currently, only pregnancies at 25% risk for SLOS are routinely subjected to testing by Dehydrocholesterol (DHC) measurement, with secondary screening of SLOS based on a finding of low E3 now being considered. However, since there are multiple causes of low maternal E3 levels, DHC measurement in amniotic fluid or villus tissue currently remains necessary for confirming diagnosis of SLOS. Unfortunately, these methods involve the analysis of compounds which are found in substantial quantities in both normal and SLOS affected patients, making the incidence of false positives higher than may be reasonably acceptable. False positives are particularly intolerable where a fetal diagnosis of SLOS may result in the mother's decision to abort. Also, these procedures are invasive in nature, making the diagnostic testing of DHC levels, in some cases, expensive, cumbersome, impractical, and even dangerous to the fetus and mother.
Thus, there is a need to develop a sensitive, non-invasive prenatal diagnostic test utilizing a analyte specific for SLOS, which can be performed early on in gestation and which is associated with a very low possibility of false positives so as to provide a reliable diagnosis of SLOS.